The treatment of disseminated cancer is a problem for clinical and veterinary medicine. The treatment regimens available today include surgery, radiation, chemotherapy, and immunotherapy. Surgery often fails due to tumor tissue which is unrecognized and not removed. Radiation and chemotherapy also fail, and the side-effects of the treatments often decrease the quality of life of the patients.
The major benefit offered by immunotherapy is that it is not generally associated with the side effects of surgery, radiation or chemotherapy. In three recent studies using dendritic cell immunotherapy in patients with cancer, minimal to no side effects were reported (Hsu, et al. Nature Medicine, 1996 2:52-58; Murphy, et al. The Prostate, 1996 29:371-380; Nestle, et al. Nature Medicine, 1998, 4 (3):328-332).
Immune therapy for cancer has been employed for many years. One of the first immune treatments was a mixed bacterial vaccine. More recently, mixtures of irradiated malignant melanoma cells have been used to induce immune responses in patients with malignant melanoma, which increased survival in several patients (Morton, et al. Ann. Surg. (1992) 216:463-482).
Newer immunotherapeutic strategies are directed toward enhancing T-lymphocyte specific immune responses toward tumors. Some strategies use genetically altered tumor vaccines, and others use non-altered antigen presenting cells to present tumor antigens to lymphocytes.
Antigen presenting cells (APCs) are naturally occurring cells which have the capability to present “foreign” and “self” antigens (proteins) to the immune system. Effective presentation of antigen to the immune system can activate T lymphocytes to fight infection and cancer. Typically, antigens which are either a part of a protein (eg. a peptide), or whole pieces of protein are co-cultured with and transferred to the APCs (Cohen P. A. et al 1994 Cancer Res 54:1055-1058), a method commonly referred to as “pulsing.”
The dendritic cell (DC) is one type of antigen presenting cell. DCs are present in small numbers in most tissues including skin, liver, lung, spleen, blood, lymphoid organs, peripheral blood, and bone marrow (Hsu F J, et al. Nature Med, 1996 2:52-58).
DCs pulsed with proteins are capable of presenting processed antigen to lymphocytes for days. After being pulsed with tumor antigen, autologous DCs have been re-infused into patients, and induced partial tumor regression in some prostate tumor patients, and lymphoma patients (Murphy G, et al. The Prostate, 1996 29:371-380, Hsu F J, et al. Nature Med, 1996 2:52-58).
Protocols have been developed to isolate and purify DCs from human blood. Three approaches used to date include:                1. Isolating bone marrow precursor cells from the blood, and stimulating them to differentiate into DCs and grow in large numbers (Romani N, et al 1994 J Exp Med, 180:83-93; Romani N et al. 1996 J Immunol Methods; 196(2):137-51 and; Reddy et al. 1997, Blood; 90(9):3640-6).        2. Collecting precommitted DCs from peripheral blood (Greudenthal P. S. et al 1990 Proc Natl Acad Sci 87:7698-7702; Mehta-Damani, et al. 1994 J Immunol 153:996-1003; Thomas R. et al. 1993 J. Immunol 151:6840-6852; and Cohen P A et al, 1997, U.S. Pat. No. 5,643,786).        3. Inducing conversion of leukapheresed monocytes using calcium ionophore (U.S. Pat. No. 5,643,786 July 1997 Cohen).        
The use of the proper tumor antigen to pulse DCs can predict the success of DC-based immunotherapy. In a recent study (Murphy G. et al. The Prostate, 1996 29:371-380) prostate cancer patients were treated with dendritic cells pulsed with an HLA-A0201 (a subset of HLA-A2 family)-specific prostate-specific membrane antigen peptide. The only patients who responded favorably to the treatment were those who expressed the HLA-A2 antigen. In another study of DC therapy in patients with B-cell lymphoma (Hsu F J et al. Nature Med, 1996 2:52-58), the antigens used to pulse DCs were idiotype proteins derived from cell fusion techniques using tumor biopsies as starting materials. Both treatment methods have shortcomings. In the case of the prostate tumor treatment, the antigen is useful only in HLA-A2+ patients. In the lymphoma treatment, the process of isolation of biopsy tumor material and cell-fusion techniques are both difficult and may be impossible in many patients. Use of a singular priming antigen to pulse the DCs could also lead to escape variant tumor cell types.
Many tumor associated antigens have been identified, and an epitope of at least one tumor antigen has been used successfully to pulse dendritic cells which resulted in partial responses in prostate cancer patients (Murphy et al. The Prostate, 1996 29:371-380). Some are used as tumor markers and rising levels can be indicative of disease progression.
Very high molecular weight (>1,000,000 daltons) tumor associated antigens have been found in the urine of 94.7% of patients with many types of cancer (Rote N S et al. 1980 Int. J. Cancer 26:203-210). Other high molecular weight tumor associated molecules have been found in the urine of cancer patients. Researchers (Chawla R K et al. 1977, Cancer Res 37:873-878) reported the presence of a glycoprotein (51,000 to 59,000 daltons) in the urine of 64% of colon cancer patients which could not be detected in the urine of non-cancer patients, but was found in the urine of 15-50% of patients with other various advanced malignancies. Another group found a >100,000 daltons molecular weight tumor-associated antigen in the urine of 65.4%-71.4% of colon cancer patients which was not detectable in 90% of healthy volunteers (Finck S J et al. 1982. J. Surg. Oncology 21:81-86). A tumor associated antigen having a molecular weight of 590,000-620,000 daltons was found in the urine of 68% of melanoma patients as opposed to 5% of normal controls (Euhus D M et al. 1989, J Clin Lab Anal 3:184-190). Prostate Specific Antigen (PSA), a 34,000 dalton molecular weight glycoprotein has been found in the urine of prostate cancer patients (deVere White R W, et al, 1992; J Urol. 147:947-951). Carcinoembryonic antigen (CEA), a high molecular weight (200,000 to 240,000 daltons) tumor marker associated with gastrointestinal tumors, has also been found in the urine of cancer patients (Halberg F, et al. 1995 In Vivo July-August; 9 (4):311-4).
Another protein (24,000 daltons) that is capable of inducing cachexia in mice has recently been found in the urine of cancer patients (Cariuk P, et al. 1997 Br J Cancer; 76:606-13). The antigen was not present in the urine of normal subjects, patients with weight loss from conditions other than cancer, or from cancer patients who were weight stable or with low weight loss (1 kg month(−1)). The antigen was present in the urine from subjects with carcinomas of the pancreas, breast, lung and ovary. The protein-induced cachexia in mice can be reversed by the administration of a monoclonal antibody to the protein to the mice.
The above list of tumor associated antigens is not complete. Other tumor associated antigens are being discovered yearly. Identification, purification, and characterization of these antigens is costly and time consuming. Until now, no tumor associated antigen with 100% specificity and sensitivity has been identified.
The ability of a high molecular weight isolate of autologous urine to stimulate anti-tumor and anti-cachexia responses was studied herein. In one embodiment the effects of dendritic cells that had been pulsed (by co-culturing and by induction of pinocytic inclusion through osmotic lysis) with a high molecular weight isolate of autologous urine (without identification, purification, or characterization of tumor associated antigens) from humans with metastatic disease was studied. As part of this investigation, it was discovered that the injection of dendritic cells pulsed with a high molecular weight isolate of urine from humans with metastatic disease did not cause any side-effects, including autoimmune diseases, when reinfused into the patient. It was also found that these dendritic cells were capable of:                1. Inducing T lymphocyte mediated cytotoxicity of human tumor cells in vitro;        2. Inducing T lymphocyte proliferation in vitro;        3. Inducing lymphocyte stimulation detected by increased interferon-gamma production in vitro; and,        4. Halting cachexia in a patient with metastatic prostate carcinoma when intravenously infused.        5. Shrinking the tumor of a patient with a large intracranial neuroblastoma.        